This project determines the structure of neuronal and glial cytoplasm, particularly as it pertains to axoplasmic transport, and the organization of cytoplasm. Cultured myocytes grown on grids, frozen, freeze-substituted, and examined directly at high voltages in an electronmicroscope have a cytoplasmic ground substance consisting of fine filaments instead of a microtubular meshwork, and distinct cytoplasmic domains characterized by organelle movements along microtubules. Microtubules isolated from the axoplasm of the squid giant axon continue to support movements of organelles for many hours. However, actin filaments in other cells support similar movements of organelles. A protein translocator responsible for these organelle movements has been characterized, a 600 KD protein with 110 KD and 60-65 KD doublet peptides. This protein induces beads to move along purified microtubules in the presence of ATP. When a monoclonal antibody column (directed towards the 110 Kd subunit) is used to purify this translocator it has the same peptide components and supports similar movements. Based on its size, pharmacological properties, and asymetrical shape seen by molecular shadowing, this translocator protein belongs to a new class of motility protein, which we call kinesin. Kinesin appears to be of general significance in cellular motility. The organelle movements induced by kinesin are always directed towards the plus ends of microtubules, a direction corresponding to anterograde axonal transport. However, brain extracts stripped of kinesin with monoclonal antibody induce bead movement in the retrograde direction. Our current efforts are concentrated on purifying the translocator for retrograde movements and on determining whether organelles selectively bind translocators in order to explain how they are selected for anterograde or retrograde transport.